The long-term goal of this work is to understand how nuclear protein TOX regulates development and function of the immune system. This protein's amino acid sequence is 94% identical between mice and humans, suggesting a highly conserved function. Dr. Kaye's laboratory has previously demonstrated that many cell types of the immune system fail to develop in TOX-deficient mice, including CD4 T lymphocytes, natural killer (NK) cells, and a specialized cell type that is required for the lymph node organogenesis. This striking biology adds particular significance to this study. We focus here on the TOX DNA binding HMG-box domain and its role in regulating gene expression during NK cell development. This is a highly evolutionarily conserved region of the protein, and one that defines the four-member TOX subfamily of proteins. NK cells, innate immune cells with cytolytic activity, play important roles in anti-viral and tumor surveillance immune functions. Understanding the regulation of NK cell development has significance for potential enhancement of immune reconstitution following bone marrow transplantation and for development of cell-based immunotherapies for cancer. The major aims of this work are (1) to test a novel hypothesis that the TOX HMG-box domain undergoes conformational change upon binding to DNA, based on the crystal structure of this domain as determined by us, (2) to determine how targeted mutations of this protein domain influence DNA binding, and (3) to determine how such mutations alter the function of the protein. We have a two-pronged approach to assess function of the protein as a regulator of gene expression. First, we have developed an innovative assay that relies on the ability of TOX to upregulate expression of a cell surface marker in an easily transfectable cell line, enabling a relatively simple quantitative assay for function. Second, we will use bone marrow progenitor cells deficient in TOX to measure restoration of NK cell development, and gene expression, upon expression of mutant forms of the TOX protein. Together, these studies will provide key insights into structure-function relationships of this protein as it relates NK cell development and form the basis for future development of conformation- dependent small molecule inhibitors or activators of the TOX-family of proteins as probes and therapeutics.